Statistical optimization for enhanced bacoside A production in plant cell cultures of Bacopa monnieri

  • J. Leonard
  • Bishwanath Seth
  • Binod B. Sahu
  • V. R. Singh
  • Nivedita Patra
Original Article


The bioactive compound, bacoside A, has immense importance for the treatment of memory disorders and Alzheimer’s disease. Due to the growing commercial interest in the herb, Bacopa monnieri, it has been listed as highly endangered species. The present study was aimed at enhancing the production of bacoside A using an alternative technology of plant cell suspension culture. Initial experiments of docking simulations using bacoside A showed good inhibition of acetyl cholinesterase (binding energy value of − 20 kcal/mol), when comparison was made with other phytocompounds and the synthetic drug for Alzheimer’s disease. In vitro experiments established that B. monnieri cell suspension culture can be developed in Murashige and Skoog medium containing containing 0.1 mg/L benzylaminopurine and 0.5 mg/L naphthalene acetic acid. Plackett–Burman studies predicted that the most effective factors for maximum biomass production were inoculum size (t-value of 4.87), sucrose concentration (t-value of 0.25) and KH2PO4 concentration (t-value of 0.007). The nitrate to ammonium ratio (t-value of − 0.42) did not have significant effect on the cell suspension biomass. The optimum concentration of the crucial variables obtained from a central composite design were—inoculum size of 2 g/L, sucrose concentration of 30 g/L and KH2PO4 concentration of 1.24 mM in one-sixth strength MS medium. The best model for optimum production of biomass and bacoside A was experimentally verified and the correlation between the predicted and actual values was found to be 99% for biomass and 94% for bacoside A production. The experimental results have been discussed in the present work.


Bacopa monnieri Benzylaminopurine Naphthalene acetic acid Bacoside A Central composite design 



The authors are thankful for the elite plant material supply from CIMAP Lucknow. The financial support by MHRD (India) for pursuing PhD (J. Leonard) and M.Tech (Bishwanath Seth) is acknowledged by two of the authors. The authors also thankfully acknowledge Life Science department (NIT Rourkela) for providing the HPLC facility which was funded by DST (FIST), India [File number: SR/FST/LSI-025/2014].

Author contributions

The docking study, development of cell culture, standardization of HPLC method and PB studies have been performed by JL. BS performed the RSM experiments, experimental validation and analysis of results. BBS and VRS were the collaborators and provided their resources for carrying out this work. NP has been the thesis supervisor for JL and BS in this research work.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Aguiar S, Borowski T (2013) Neuropharmacological review of the nootropic herb Bacopa monnieri. Rejuvenation Res 16:313–326. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bagherieh-Najjar M, Nezamdoost T (2016) Optimization of shikonin production in Onosma dichroantha callus using response surface methodology. Plant Cell Tissue Organ Cult 126:399–409. CrossRefGoogle Scholar
  3. Bansal M, Sudhakara Reddy M, Kumar A (2017) Optimization of cell growth and bacoside-A production in suspension cultures of Bacopa monnieri (L.) Wettst. using response surface methodology. In Vitro Cell Dev Biol-Plant. Google Scholar
  4. Benilova IV, Arkhypova VM, Dziadevych SV, Jaffrezic-Renault N, Martelet C, Soldatkin OP (2006) Kinetic properties of butyrylcholinesterases immobilised on pH-sensitive field-effect transistor surface and inhibitory action of steroidal glycoalkaloids on these enzymes. Ukr Biokhim Zhurnal 78:131–141Google Scholar
  5. Bulgakov VP, Tchernoded GK, Mischenko NP, Khodakovskaya MV, Glazunov VP, Radchenko SV, Zvereva EV, Fedoreyev SA, Zhuravlev YN (2002) Effect of salicylic acid, methyl jasmonate, ethephon and cantharidin on anthraquinone production by Rubia cordifolia callus cultures transformed with the rolB and rolC genes. J Biotechnol 97:213–221CrossRefPubMedGoogle Scholar
  6. Cataldo DA, Haroon M, Schrader LE, Youngs VL (1975) Rapid colorimetric determination of nitrate in plant-tissue by nitration of salicylic-acid. Commun Soil Sci Plant Anal 6:71–80. CrossRefGoogle Scholar
  7. Chatterji N, Rastogi RP, Dhar ML (1965) Chemical examination of Bacopa monniera Wettst.: parti-isolation of chemical constituents. Indian J Chem 3:24–29Google Scholar
  8. Deepak M, Sangli GK, Arun PC, Amit A (2005) Quantitative determination of the major saponin mixture bacoside A in Bacopa monnieri by HPLC. Phytochem Anal 16:24–29CrossRefPubMedGoogle Scholar
  9. Fadel D, Kintzios S, Economou AS, Moschoupoulou G, Constantinidou HI (2010) Effect of different strength of medium on organogenesis, phenolic accumulation and antioxidant activity of spearmint (Mentha spicata L.). Open Hortic J 3:31–35CrossRefGoogle Scholar
  10. Goleniowski M, Trippi VS (1999) Effect of growth medium composition on psilostachyinolides and altamisine production. Plant Cell Tissue Organ Cult 56:215–218CrossRefGoogle Scholar
  11. Hanchinal VM, Survase SA, Sawant SK, Annapure US (2008) Response surface methodology in media optimization for production of β-carotene from Daucus carota. Plant Cell Tissue Organ Cult 93:123–132. CrossRefGoogle Scholar
  12. Hand C, Reed BM (2014) Minor nutrients are critical for the improved growth of Corylus avellana shoot cultures. Plant Cell Tissue Organ Cult 119:427–439. CrossRefGoogle Scholar
  13. Hegazi G, Taha H, Sharaf AMM, Elaish SR (2017) Enhancing in vitro production of bacoside A from Bacopa monnieri using precursor and elicitors feeding. J Basic Appl Sci Res 7:27–35Google Scholar
  14. Heinrich M, Teoh HL (2004) Galanthamine from snowdrop—the development of a modern drug against Alzheimer’s disease from local Caucasian knowledge. J Ethnopharmacol 92:147–162CrossRefPubMedGoogle Scholar
  15. Jimbo D, Kimura Y, Taniguchi M, Inoue M, Urakami K (2009) Effect of aromatherapy on patients with Alzheimer’s disease. Psychogeriatrics 9:173–179CrossRefPubMedGoogle Scholar
  16. Jung HK, Eun JC, Hoon O (2005) Saponin production in submerged adventitious root culture of Panax ginseng as affected by culture conditions and elicitors. Asia Pac J Mol Biol Biotechnol 13:87–91Google Scholar
  17. Kennedy DO, Wightman EL, Okello EJ (2010) Medicinal plants, phytochemicals and Alzheimer’s disease. In: Martinez A (ed) Emerging drugs and targets for Alzheimer’s disease. Royal Society of Chemistry, London, pp 269–301CrossRefGoogle Scholar
  18. Majumdar S, Garai S, Jha S (2011) Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of Bacopa saponins in transformed calli and plants. Plant Cell Rep 30:941–954CrossRefPubMedGoogle Scholar
  19. Miller GH (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  20. Montgomery DC (1997) Design and analysis of experiments, 4th edn. Wiley, New YorkGoogle Scholar
  21. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  22. Murphy J, Riley JP (1962) A modified single solution method for determination of phosphate in natural systems. Anal Chem Acta 27:31–36CrossRefGoogle Scholar
  23. National Center for Biotechnology Information. PubChem Compound Database (2017) CID = 92043183, Accessed 29 Nov 2017
  24. National Medicinal Plants Board (NMPB) and Technology Information Forecasting and Assessment Council (TIFAC) (2007) Department of Science and Technology, Government of India. (
  25. Patra N, Srivastava AK (2014) Enhanced production of artemisinin by hairy root cultivation of Artemisia annua in a modified stirred tank reactor. Appl Biochem Biotechnol 174:2209–2222CrossRefPubMedGoogle Scholar
  26. Patra N, Srivastava AK (2016) Artemisinin production by plant hairy root cultures in gas- and liquid-phase bioreactors. Plant Cell Rep 35:143–153CrossRefPubMedGoogle Scholar
  27. Patra N, Sharma S, Srivastava AK (2012) Statistical media optimization for enhanced biomass and artemisinin production in Artemisia Annua hairy roots. In: Khemani LD, Srivastava MM, Srivastava S (eds) Chemistry of phytopotentials: health, energy and environmental perspectives. Springer-Berlin, Heidelberg, pp 173–176.
  28. Prakash G, Srivastava AK (2005) Statistical media optimization for cell growth and azadirachtin production in Azadirachta indica (A. Juss) suspension cultures. Process Biochem 40:3795–3800. CrossRefGoogle Scholar
  29. Rahman LU, Verma PC, Singh D, Gupta MM, Banerjee S (2002) Bacoside production by suspension cultures of Bacopa monnieri (L.) Pennell. Biotechnol Lett 24:1427–1429CrossRefGoogle Scholar
  30. Ramasamy S, Chin SP, Sukumaran SD, Buckle MJC, Kiew LV, Chung LY (2015) In silico and in vitro analysis of bacoside A aglycones and its derivatives as the constituents responsible for the cognitive effects of Bacopa monnieri. PLoS ONE 10:e0126565CrossRefPubMedPubMedCentralGoogle Scholar
  31. Raval KN, Hellwig S, Prakash G, Ramos-Plasencia A, Srivastava A, Buchs J (2003) Necessity of a two-stage process for the production of azadirachtin-related limonoids in suspension cultures of Azadirachta indica. J Biosci Bioeng 96(1):16–22CrossRefPubMedGoogle Scholar
  32. Singh M, Chaturvedi R (2012) Statistical optimization of media for enhanced azadirachtin production from redifferentiated zygotic embryo cultures of neem (Azadirachta indica A. Juss.). In Vitro Cell Dev Biol-Plant 48:92–98. CrossRefGoogle Scholar
  33. Srivastava S, Srivastava AK (2007) Hairy root culture for mass-production of high-value secondary metabolites. Crit Rev Biotechnol 27:29–43CrossRefPubMedGoogle Scholar
  34. Trejo-Tapia G, Arias-Castro C, Rodríguez-Mendiola M (2003) Influence of the culture medium constituents and inoculum size on the accumulation of blue pigment and cell growth of Lavandula spica. Plant Cell Tissue Organ Cult 72:7–12CrossRefGoogle Scholar
  35. Vijaykumar M, Vijaykumar R, Stephen R (2010) In vitro propagation of Bacopa monnieri L.—a multipurpose medicinal plant. Indian J Sci Technol 3:781–786Google Scholar
  36. Wang R, Yan H, Tang X (2006) Progress in studies of huperzine A, a natural cholinesterase inhibitor from Chinese herbal medicine1. Acta Pharmacol Sin 27(1):1–26CrossRefPubMedGoogle Scholar
  37. Weathers PJ, DeJesus-Gonzalez L, Kim YJ, Souret FF, Towler MJ (2004) Alteration of biomass and artemisinin production in Artemisia annua hairy roots by media sterilization method and sugars. Plant Cell Rep 23:414–418CrossRefPubMedGoogle Scholar
  38. Wu C-H, Dewir YH, Hahn E-J, Paek K-Y (2006) Optimization of culturing conditions for the production of biomass and phenolics from adventitious roots of Echinacea angustifolia. J Plant Biol 49:193CrossRefGoogle Scholar
  39. Yadav J, Balabantaray S, Patra N (2017) Statistical optimization of fermentation conditions for the improved production of poly-β-hydroxybutyrate from Bacillus subtilis. Chem Eng Commun. Google Scholar
  40. Zhang Z-Y, Zhong J-J (2004) Scale-up of centrifugal impeller bioreactor for hyperproduction of ginseng saponin and polysaccharide by high-density cultivation of panax notoginseng cells. Biotechnol Prog 20:1076–1081CrossRefPubMedGoogle Scholar
  41. Zhao D, Xing J, Li M, Lu D, Zhao Q (2001) Optimization of growth and jaceosidin production in callus and cell suspension cultures of Saussurea medusa. Plant Cell Tissue Organ Cult 67:227–234. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  1. 1.Department of Biotechnology and Medical EngineeringNational Institute of Technology (NIT)RourkelaIndia
  2. 2.Department of Life ScienceNITRourkelaIndia
  3. 3.Central Institute of Medicinal and Aromatic Plants (CIMAP), P.O. CIMAPLucknowIndia

Personalised recommendations